The measurement of N-protein activity in plasma membranes: the comparison of assay methods by reconstitution of cyc- membranes and cholera toxin-catalyzed ADP ribosylation

Abstract

The relationship between the assay values of the activities of the stimulatory guanine nucleotide-binding regulatory component (N-protein) of adenylate cyclase by two different methods: reconstitution of plasma membranes of cyc- S49 cells and ADP ribosylation catalyzed by cholera toxin, have not been fully elucidated yet. In the present study, the reconstitution and ADP ribosylation assay methods were utilized, and the relationship between the assay values of the N-protein activities of the erythrocyte membranes from normal subjects and patients with pseudohypoparathyroidism type I (PHP-I) measured by the two methods was investigated. When Lubrol extracts of human erythrocyte membranes were reconstituted with cyc- membranes, the rate of cyclic AMP synthesis reached a constant rate after incubation of 80 minutes at 30 degrees C. This reaction depended on the concentrations of cyc- membranes and Lubrol extracts of the erythrocyte membranes. N-protein activity in the erythrocyte membranes of normal subjects and PHP-I patients assayed by reconstitution of cyc- membranes (mean +/- SD, expressed by % of pooled standard preparation) was 100.1 +/- 13.5 (n = 29) and 82.3 +/- 28.0 (n = 19) respectively. The cholera toxin-catalyzed transfer of [32P]ADP ribose from [32P]NAD to the 42,000-dalton peptide subunit of human erythrocyte N-protein reached a plateau after incubation of 10 minutes at 30 degrees C. This reaction depended on the concentrations of cholera toxin, NAD, and the erythrocyte membranes. N-protein activity in the erythrocyte membranes from normal subjects and PHP-I patients assayed by cholera toxin-catalyzed ADP ribosylation (pmol/mg of protein) was 1.33 +/- 0.20 (n = 7) and 1.15 +/- 0.43 (n = 5) respectively. The correlation between assay values of erythrocyte N-protein activity in PHP-I patients by reconstitution of cyc- membranes (X, % of pooled standard) and cholera toxin-catalyzed ADP ribosylation (Y, % of pooled standard) was recognized as a linear regression: Y = 0.77X + 16.8 (r = 0.981, P less than 0.001). It is thus concluded that the activity of N-protein in human erythrocyte membrane can be assayed by the ADP ribosylation method more simply than by the method of reconstitution of cyc- membranes, with highly close correlation between the values determined by these two methods.